Secretion of Ricinus communis glyceraldehyde-3-phosphate dehydrogenase by Escherichia coli.
A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer-membrane protein A to the 5'-end of a glyceraldehyde-3-phosphate dehydrogenase cDNA from Ricinus communis. When expressed in E. coli, the fusion protein was secreted by the normal membrane-potential-dependent pathway. Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol. Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane. The protein could not be released from the membrane by washing with alkaline sodium carbonate. Radioactivity from [U-14C]-palmitate was incorporated into the heterologous protein. These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid-modification site, and the combination of a signal sequence plus a lipid-modification sequence results in specific targeting to the bacterial outer membrane.
Hekman
W E
WE
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Dennis
D T
DT
Miernyk
J A
JA
D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010947:Plants, Toxic; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012278:Ricinus / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases